299 High efficiency and safe base editor correction of recurrent COL7A1 mutations underlying dominant dystrophic epidermolysis bullosa

      Dominant dystrophic epidermolysis bullosa (DDEB) is an inherited blistering skin disease caused by heterozygous mutations in COL7A1, which encodes type VII collagen (C7), the major component of anchoring fibrils that maintain the integrity of the dermal-epidermal junction. Most COL7A1 mutations in DDEB are single nucleotide variants, resulting in glycine substitutions within the C7 triple helix. Functionally, such mutations cause impaired assembly or secretion of C7 and result in dominant-negative interference through faulty C7 assembly of wild-type and mutant chains into anchoring fibrils. Adenine base editing (ABE) mediates A-T to G-C base pair changes and is particularly suited to correcting G>A mutations, which account for over 60% of DDEB cases. Here, we used base editing to target three of the most common recurrent COL7A1 mutations (all in exon 73) which result in DDEB. Patient derived fibroblasts carrying either c.6082 G>A (p.Gly2028Arg), or c.6100 G>A (p.Gly2034Arg), or c.6127 G>A (p.Gly2043Arg) in of COL7A1 were edited using ABE8e and a single guide RNA (sgRNA) targeting each mutation. Electroporation and lipid nanoparticle delivery methods of the base editing tools into the cells were tested, with lipofection yielding higher editing efficiency for all these mutations. Notably, we achieved 100% base correction of the c.6100 G>A mutation using lipofection, with no bystander mutations present. Western blot analysis of the 100% edited cells demonstrated less intracellular retention and normal levels of C7 secretion into the extracellular matrix, comparable to wild-type fibroblasts. Base editing is a promising approach to correct the dominant-negative interference phenotype of DDEB. Using lipofection, which is an in vivo compatible method of base editor delivery, makes the results of this study especially relevant for future clinical translation